Current storage of fresh human platelets in liquid form (i.e., as platelet-rich concentrate in residual plasma) is limited in the following ways:
1) Platelets in liquid suspension (platelet concentrate) are generally not stored refrigerated as they suffer from a well-documented "storage lesion" involving loss of clotting function and in vivo viability (refer to AABB Technical Manual, 10th edition, 1990, pp.51-52). Platelet concentrates stored refrigerated are limited to a 72 hour shelf life due to the rapidity of the storage decay. The method used by most blood banks is to store platelet concentrate at room temperature (20-24 deg. C.) with continuous agitation by mechanical rocking. Agitation is essential at these temperatures to prevent cell aggregate formation. Currently regulations of the U.S. Food and Drug Administration (FDA) limit room temperature storage to 5 days. PA1 2) The quality of room temperature stored platelet concentrates, as measured by aggregometry and other in vitro measures, decays after the first 24 hours of storage to some 75-85% of fresh values. Further deterioration occurs by five days of storage, such that the therapeutic value is only about 30% of fresh cells (i.e., the clotting efficacy of infused 5 day-old concentrate in patients, as measured by their bleeding time reduction, is only some 30% of a comparable dose of fresh platelets). PA1 3) FDA limits room temperature storage of platelet concentrates to five days due to the threat of bacterial growth in the nutrient rich plasma concentrate. Contamination of platelet concentrates by Yersinia bacteria has been associated with deaths due to platelet transfusion. PA1 4) The short shelf life means that some 25% of platelet concentrates collected by U.S. blood banks become unusable by outdating. This is a loss of a valuable voluntary resource.
The current useful method of storage of platelet concentrates in frozen form uses a 5% DMSO storage method developed by Schiffer et al (1983) Annals N.Y. Acad. Sci. 411, 161-169. This requires that the cells be stored at -120 deg. C. in the vapor phase of liquid nitrogen. Most blood banks do not use this method due to the expense of liquid nitrogen storage, plus the impracticality of shipping in liquid nitrogen. (See AABB Technical Manual, 1990, pp. 100-101 for review of blood cell freezing literature). Valeri has reported a 6% DMSO method that allows storage at temperatures as high as -65 deg. C., (referenced in the AABB Manual p. 102), a temperature compatible with ultra-low mechanical freezers. These freezers are also very expensive and have limited storage capacity.
An accepted shelf life for frozen platelets in 5% DMSO is 3 years. In actual clinical use, a frozen-thawed DMSO unit of platelets will normally exhibit about 50-60% of the clotting efficacy seen with a fresh platelet concentrate unit (i.e., usually two units of frozen thawed platelets are needed to achieve the same effect in vivo as one fresh unit).
It should be noted that FDA has not approved any DMSO platelet frozen storage method due to controversy over the efficacy and possible toxicity of DMSO (usually the frozen cells are thawed and infused directly, along with the 5% DMSO). Thus, U.S. blood banks today only freeze and infuse platelets with the approval of their medical review boards (i.e., interstate transport is prohibited).
Platelets are collected at most blood collection facilities, ranging from large regional blood centers (over 100,000 donations per year) to local hospital blood banks. Platelets can only be stored for 5 days as a platelet-rich plasma fraction at room temperature according to current FDA regulations. Although platelets can be frozen at -80 degrees for up to two years, the expense of freezing has discouraged frozen storage among civilian entities. The short shelf life is a major impediment to directed pre-deposit of plateletpheresis units.
A second issue problem is the transport of frozen platelets, which presents difficulties in container design, especially for air shipment. There is thus a need for lyophilized platelets which can be stored, shipped and reconstituted for therapeutic use.
Platelets play a vital role in blood clot formation and platelet transfusions are administered to arrest an existing bleeding condition or to prevent an imminent loss of blood. Often platelet transfusions are indicated when the patient cannot mobilize his own platelet reserves, as a result of standard chemotherapy treatments or diseases that cause low levels of circulating platelets or loss of normal platelet function. The American Red Cross (ARC) in 1987 reported a total of 4,500,000 transfusions of random donor platelet units and 350,000 transfusions of plateletpheresis units during calendar year 1986 in the United States. Some 245,000 patients received platelet transfusions during this period. The average patient receiving random donor units isolated from whole blood donations required 6 platelet units per transfusion event and also required 6 separate transfusions per year. The high rate of transfusions reflects the need for multiple transfusions for cancer patients undergoing aggressive chemotherapy or bone marrow transplantation as an adjunct to chemotherapy.
Multiple platelet transfusions can cause immune sensitization and rejection, especially when random donor units are used. This sensitization reaction, combined with improved plateletpheresis methods, has spurred demand for directed-donor plateletpheresis units. Plateletpheresis selectively isolates platelets from whole blood and returns the depleted blood to the donor's arm. This allows collection of concentrated platelet units from a single donor, as often as once per week. Each plateletpheresis unit contains the equivalent of 5-6 units obtained from donated whole blood (random donor units). Plateletpheresis allows more closely matched donors, such as close relatives, to donate and minimize the risk of sensitization. The use of concentrated plateletpheresis units also reduces the patient's exposure to numerous random donors, which reduces both sensitization risk and exposure to viral contaminants (a chemotherapy patient may require 60 platelet units during the course of treatment, and may be exposed to over 50 random donors).
Platelets are prescribed to control massive bleeding events before or during cardiopulmonary bypass surgery, and organ transplants. Unlike red cells, the normal human body possesses platelet reserves that can be mobilized rapidly if blood is lost. As a result, the U.S. Department of Health and Human Services advises that platelets not be wasted on prophylactic transfusions for routine surgery or even most trauma situations (patients can cope with a major bleed of 20 units by mobilizing platelets provided red blood cells are administered).
Bone marrow transplants designed to restore marrow destroyed by chemotherapy accounts for the single most intensive use of platelet transfusions. Most patients recovering from chemotherapy require at least three weeks (and sometimes months) before the new bone marrow graft can begin to produce platelets. During this period circulating platelet levels are maintained by transfusions, which must be frequent due to the short 5 day circulation lifetime of normal platelets.
The present invention provides a method utilizing a buffer for platelets which allows frozen storage at high temperatures (i.e., in the range of -20 deg. C.). This temperature range is easily achieved by conventional mechanical freezers such as chest freezers now used for food storage. The present invention enables long term frozen storage, which will obviate the short shelf life of room temperature or refrigerated storage, as well as the threat of bacterial growth in room temperature stored liquid concentrates. The prohibitive costs of liquid nitrogen frozen storage or ultra-low temperature mechanical freezers are also eliminated, and the use of DMSO is eliminated.
The present invention provides a method for freezing (and freeze-drying) cells or cell-like materials, including platelets, by the use of polymer glass transition theory to determine an effective cryopreservation for natural cells suspended in an aqueous environment. As reviewed by Levine and Slade (1987) Water Science Reviews (F. Franks, ed.) Vol. 3, pp. 79-185, Cambridge University Press, in aqueous systems comprising many natural and synthetic polymers, water acts as a plasticizer that affects the glass-to-rubber transition temperatures (Tg') of the aqueous-polymer system. This area of research has had a significant impact on the design of polymeric food materials (such as those based on starches), since the raising or lowering of Tg' can confer unique advantages for processing or storage stability of a material.
A working definition of a glass (Levine and Slade, 1987) is that it comprises a mechanical solid capable of resisting flow. In a typical amorphous glass the viscosity is extremely high, in the range of 10.sup.11 to 10.sup.14 Pa-s at the glass transition temperature (Tg). The Tg value, as illustrated in FIG. 2.4 of Slade and Levine (1987) occurs at the transition from the glassy fluid to the rubbery fluid state for systems comprising glassy or partially crystalline polymers. Below Tg, in the glass phase, the high viscosities preclude molecular diffusion and hence chemical reactions (which can lead to product spoilage) cannot proceed at significant rates. From this same figure another important point can be seen: that once the product temperature exceeds Tg, viscous flow can occur in the rubbery state, allowing for molecular diffusion and chemical reaction rates to proceed with exponential kinetics (WLF kinetics).
As a practical example, this explains why certain frozen and perishable biological products, such as human blood cells, must be stored within carefully defined temperature ranges (American Association of Blood Banks Technical Manual, 10th edition, 1990, chapter 5, pp. 91-103). Currently accepted blood cell freezing solutions containing glycerol or dimethylsulfoxide (DMSO) form glasses at very low temperatures (between -65.degree. C. and -198.degree. C.), thereby maintaining the cells in a chemically unreactive environment. However, exposure to temperatures that exceed Tg lead to poor cell viability as measured by red blood cell circulation time following freeze-thawing and transfusion or bleeding time reduction following transfusion of frozen-thawed platelets. In essence, the perishable cells can spoil even at temperatures below 0.degree. C. normally thought of as "freezing temperatures" because the cryoprotective media have Tg values much lower than the -20.degree. C. range commonly achieved by standard kitchen freezers.
Many of the fundamental principles of glass transition theory have derived from physical chemical studies in single component systems (see Levine and Slade, 1987) capable of forming a homogeneous "pure" glassy phase at the glass transition temperature (Tg) characteristic of that component, and in multicomponent systems. The present invention, however, provides multi-component aqueous cryopreservative systems that at appropriate temperatures form partially crystalline mixtures of water ice with interspersed regions of a separate amorphous glass phase. As defined in Pikal (1990), BioPharm, September-October 1990, the glass transition temperature of the amorphous phase in a partially crystalline aqueous system will be defined as Tg', to distinguish this particular phase from the glass transition temperatures of individual buffer components (i.e., the component Tg values).